The phenomenon of mitochondrial RNA editing in kinetoplastid protozoans is the most radical form of post transcriptional alteration known to date. The editing of messenger transcripts in these organisms can create over half of the mRNA by uridine insertions. It allows the generation of translatable messages by creating the open reading frames as well as proper initiation and termination signals. In Trypanosoma brucei, there is a transcript specific mechanism that regulates editing during the life cycle of the parasite. However, almost nothing is known about the mechanism of RNA editing or how editing might be developmentally regulated. The overall objective of this proposal is to identify the components of the editing mechanism and to determine the pathway and requirements of their assembly into an active complex. This project will be approached using three avenues of research: 1) An analysis of the proteins and/or RNAs which specifically associate with unedited mRNA transcripts. Approaches used in analysis of spliceosomes and polyadenylation complexes will be employed as models, including: native gel electrophoresis, UV crosslinking, gel filtration and affinity chromatography. The pathway and requirement of assembly of these components into an active complex will be investigated utilizing native gel electrophoresis and two partial in vitro editing assays. 2) A determination of the sequence and secondary structure requirements for gRNA selection and use. By manipulating input pre-edited mRNAs and gRNAs in partial in vitro editing assays, the minimum sequence and 2 degree structure requirements for gRNA recognition and function will be determined. In addition, different models for how guide RNAs interact with and direct the editing process will be tested. 3) Development of a complete in vitro editing system. An assay in which a full cycle of editing can be monitored is crucial to the identification of specific editing components and to the determination of their individual functions. Therefore, considerable effort will be invested in developing a functional assay. Members of the kinetoplastida are the causative agents of African sleeping sickness, Chagas disease and leishmaniasis. There are no vaccines against these organisms and present chemotherapies are quite toxic. kRNA editing is unique to these organisms, and offers a prime target for the development of new treatments. The ability of small RNAs to dramatically increase the coding capacity of another class of RNA offers strong support for the role of RNA in the origins of life. Understanding RNA editing in these organisms may lend considerable insight to the evolution of early replication mechanisms, and has substantial implications on our understanding of the storage and regulation of genetic information.